Enzyme Mechanisms and Inhibition

Pratt & Cornely Ch 7

Other Factors

• Other factors affect enzyme activity– Temperature– pH

pH Optimum

• Determined by structural stability– Compartmentalization

• Determined by active site residues– Bases must be

deprotonated– Acids must be

protonated

Mechanisms

• Two major mechanisms—any or all may be used in a given enzyme– Chemical Mechanisms: Change the pathway• Acid-base catalysis• Covalent catalysis• Metal ion catalysis

– Binding Mechanisms: Lower Activation energy• Proximity/orientation effect• Transition State Stabilization

Case Study 1: Chymotrypsin

• Well studied enzyme

• Example of how enzyme mechanism studied

• Example of types of mechanism

Catalyzes hydrolysis of amide

Enzyme Assays

• Make artificial substrate• Product formation may be followed with UV-

vis spectroscopy

Group Specific Reagents

• Chemical that reacts with a particular residue• Covalent modification• Can be used to determine especially reactive

serine-195

Affinity Label

• Chemical that has affinity for active site• Discovered active site Histidine

Pre-Steady State Kinetics

• “Burst” kinetics• Small initial

product formation followed by steady rate of product formation

• Suggests a two step mechanism

Covalent Catalysis

• Consistent with two phases• First, enzyme forms bond with substrate to

give initial burst of product• Then the catalytic cycle begins

Proposed Mechanism:Catalytic Triad

• Roles for serine, histidine, and aspartate

Catalysis

• Uncatalyzed hydrolysis of amide– Look for points of instability (high transition

states)• Acid-catalyzed hydrolysis– Stabilize places of instability– Break one high hurdle into more lower hurdles

• Enzyme-catalyzed hydrolysis

Stabilization of intermediates

• Locate high energy intermediates

• How are they stabilized?

• Oxyanion hole– H-bonds– Bond angles

Origin of Substrate Specificity

• Specificity binding pocket

• Also orients amide bond into the right location

Case Study 2: Metalloprotease

• Propose a mechanism for this protease:

Irreversible Enzyme Inhibition

• Some compounds inhibit enzymes irreversibly– Covalent linkage– Tight binding

• Major Types– Group-Specific– Affinity labels– Mechanism-based– Transition State Analog

Mechanism Based Inhibitors

• Suicide inhibitors• Selectivity• Targeting fast-

growing cells

Drug Byproducts

• Oxidation of xenobiotics by P450 enzymes• Pharmacology• Liver damage—covalent binding to cysteine

Case Study: Penicillin

• First antibiotic discovered

• Highly reactive b-lactam bond

• Works against a bacterial target not found in humans

Glycopeptide Transpeptidase

• Peptidoglycan gives Staph its structural support

• Crosslinked pentagly to D-Ala

Trojan Horse

• Penicillin mimics substrate, but does not become unbound

Transition State Analog

• Your book presents high energy intermediate analog

Designing a Transition State Analog

Binding of Transition State Analog

Case Study: Orotidine Decarboxylase

Mechanism of Catalysis

Reversible Inhibition Kinetics

• Know types of Reversible Inhibition• Know effect on kinetic parameters• Understand why• Interpret MM and L-B plots

Review: Lineweaver Burke Plot

• Analyze hyperbola• Construct linear plot• Double reciprocal

Competitive Inhibition

• Binds to same site as substrate-both cannon be bound at same time

• EI complex is inactive• Usually structurally

resembles substrate• Sulfanilamide• Draw altered MM plot

Competitive Inhibition

• Enough added substrate can outcompete inhibitor

• “Feels like…”– Same amount of

Enzyme at high [S]• Vmax unchanged

– Needs more S to bind (lowers affinity)• Km raised

Uncompetitive Inhibition

• Inhibitor binds only to [ES], not to free [E]

• [ESI] is inactive• Added substrate

increases inhibitor effect

• Glyphosate (Roundup)• Draw altered MM plot

Uncompetitive Inhibition

• Increased substrate increases inhibitor effect

• “Feels like…”– Less enzyme at high [S]• Decrease Vmax

– Enzyme has greater affinity for substrate• Decrease Km

Noncompetitive Inhibition

• Assumes simple case of inhibitor binding equally to E and ES

• [ESI] inactive• Only in case where

inhibitor binding affects chemical rate (kcat) but not binding (Km)

• Very rare• Draw altered MM plot

Noncompetitive Inhibition

• “Feels like…”– Less enzyme at all [S]• Decrease Vmax

– No effect on substrate affinity (no net shift in equilibrim)• Km(app) same as Km

Mixed Inhibition

• Like noncompetitve, but not the simple case– Inhibitor may bind E or

ES better• “Feels like…”– Less enzyme at all [S]– Overall lowering OR

raising of affinity for substrate

Mixed inhibition

Fill in the ChartInhibition Effect on KM Effect on Vmax Effect on Vmax/KM

Competitive

Uncompetitive

Noncompetitive

Mixed

Problem

• A cysteine protease was discovered that has an inhibitor called P56. Shutting down this enzyme could be a strategy for treating malaria. What type of inhibitor is P56?